The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

Abstract

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

Tes is recruited to focal adhesions. (A) Schematic representation of Tes and the GFP-tagged expression clones including the location of point mutations inactivating each LIM domain used in this study. The in vivo localization of the GFP-tagged proteins and their interacting partners based on Western blot analysis of pull-down assays are indicated. Only the interactions between LIM1-zyxin and the two halves of Tes have been shown to be direct. N.D, not determined. (B) Immunofluorescence analysis of HeLa cells reveals that GFP-Tes or endogenous Tes colocalize with paxillin at focal adhesions. Bar, 20 μm. (C) Western blot analysis with Tes antibody detects a single band of the correct predicted size in HeLa cell extracts. Molecular mass markers are indicated in kDa.

Analysis of the cellular localization of GFP-tagged Tes domains. Immunofluorescence analysis of HeLa cells expressing the indicated GFP-Tes colabeled for paxillin and phalloidin. The NH2-terminal half of Tes (Tes-N-term) is associated with actin stress fibers, lamellipodia, and focal adhesions. The COOH-terminal half of Tes (Tes-C-term) is largely observed at focal adhesions and weakly observed along stress fibers. The LIM1, but not the LIM2 (not shown) or LIM3 domain, is strongly recruited to focal adhesions and weakly recruited along stress fibers. Disruption of LIM1 (Tes-C265A) or LIM2 (Tes-C328A) (not depicted) in full-length Tes does not affect recruitment of the protein to focal adhesions but results in increased localization along stress fibers. Disruption of the LIM3 domain (Tes-C391A) results in a diffuse cytoplasmic localization. Bar, 20 μm.

Tes interacts directly with zyxin; the two halves of Tes interact with each other. (A) Western blot analysis with the indicated antibodies (left) of pull-down assays performed on HeLa cell extracts using the Ni affinity resins indicated (top). The His-Gem affinity resin (Gem) represents a negative control. The input extract (Extract) as well as the bound fraction (Bound) and supernatant (Sup) for each resin are indicated. (B) Western blot analysis with the indicated antibodies (left) of Tes immunoprecipitation assays. The input extract (Extract), anti-Tes antiserum (anti-Tes) or the preimmune serum (Pre-imm) are indicated. (C) Western blot analysis of pull-down assays performed on HeLa cell extracts using the His-LIM resins indicated (top) shows that the LIM1 domain interacts with zyxin, whereas LIM3 binds Mena and VASP. (D) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing GST-zyxin with the His-LIM resins shows that only the LIM1 domain is able to interact directly with zyxin. (E) Western blot analysis with anti-GST antibody of pull-down assays performed on E. coli soluble fraction containing the GST fusion protein (bottom) with purified His protein resins (top) demonstrates that the NH2-terminal half of Tes can interact directly with the COOH-terminal half of the molecule in vitro. (F) Western blot analysis of Ni resin pull-downs from extracts of HeLa cells cotransfected with His-Tes-C-term and GFP-Tes-N-term. The anti-GFP blot demonstrates that GFP-Tes-N-term copurifies with His-Tes-C-term but not His-Gem, indicating the halves of the molecule interact with each other in vivo.

Recruitment of Tes and VASP to focal adhesions is dependent on zyxin. (A) Immunofluorescence analysis demonstrates that endogenous paxillin, zyxin, and Tes (red) are still recruited to focal adhesions in the absence of Ena/VASP proteins in MVD7 cells. The actin cytoskeleton is visualized with phalloidin (green). Bar, 20 μm. (B) Immunofluorescence analysis of mixed populations of HeLa cells transfected with zyxin and control siRNA oligos. The left column corresponds to the zyxin signal, whereas the right column shows the indicated protein. In cells lacking zyxin (white arrowheads), there is a corresponding absence of Tes, VASP, and Mena (not depicted) but not paxillin or vinculin at focal adhesions. Bar, 20 μm. (C) Schematic representation of the conformation changes in Tes and the proteins with which it associates. Double-headed arrows indicate associations based on pull-downs or immunoprecipitations, which may not represent direct interactions. In the cytoplasm, the molecule is in a “closed” conformation but can still associate with actin and VASP/Mena. Upon “activation” by an unknown mechanism, the molecule adopts a more “open” conformation and is recruited to and/or stabilized at focal adhesions. In this “open” conformation, Tes is able to associate with α-actinin, actin, paxillin, Mena, VASP, and bind directly to zyxin.

Tes negatively regulates growth of T47D cells. (A) Phase–contrast images of T47D cells, wild-type or expressing GFP or GFP-Tes, taken from movie sequences at the times indicated. Video available at www.jcb.org/cgi/content/full/jcb.200211015/DC1. (B) Graph showing average fold increase in cell number over times indicated. Error bars represent standard deviation of three independent experiments. (C) T47D colonies in soft-agar stained with nitroblue tetrazolium after 14-d growth. (D) Graph showing number of colonies (gray) formed by T47D cells (wild-type or expressing GFP or GFP-Tes) and the number of colonies with diameter greater than 100 μm (black). Data represent mean ± standard deviation between three independent experiments. Expression of GFP-Tes in T47D cells reduces the number and size of colonies formed when compared with cells expressing GFP.